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WO1997002279A1 - Procedes et compositions permettant de moduler ou d'inhiber la telomerase - Google Patents

Procedes et compositions permettant de moduler ou d'inhiber la telomerase Download PDF

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Publication number
WO1997002279A1
WO1997002279A1 PCT/US1996/011421 US9611421W WO9702279A1 WO 1997002279 A1 WO1997002279 A1 WO 1997002279A1 US 9611421 W US9611421 W US 9611421W WO 9702279 A1 WO9702279 A1 WO 9702279A1
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Prior art keywords
telomerase
deaza
triphosphate
cells
lower alkyl
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PCT/US1996/011421
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English (en)
Inventor
Shih-Fong Chen
Bradford E. Windle
Terace M. Fletcher
Ira M. Maine
Sean M. Kerwin
Miguel Salazar
Blain Mamiya
Makoto Wajima
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Ctrc Research Foundation
Board Of Regents, The University Of Texas System
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Priority to AU64859/96A priority Critical patent/AU6485996A/en
Publication of WO1997002279A1 publication Critical patent/WO1997002279A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/14—Pyrrolo-pyrimidine radicals

Definitions

  • the present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions useful in modulating or inhibiting human telomerase activity. In certain embodiments, the invention concerns the use of these agents in treatment of proliferative cell disorders, particularly for cancers whose proliferation is determined by processive telomerase activity.
  • Telomeres play an important role in chromosome organization and stability.
  • telomere activity is not detected in normal somatic cells leading to the implication of telomerase in cancer and the impetus to develop agents that selectively target telomerase activity.
  • telomeres provide stability to the chromosomes.
  • Tumor cells have shortened telomeres, but they also possess greatly elevated levels of the enzyme telomerase to overcome the end-replication problem, while normal cells do not.
  • telomerase is an attractive target for new anti-cancer agents because of the expected selectivity for neoplastic cells.
  • Telomeres consist of simple DNA repeats at the end of eukaryotic chromosomes and the proteins that bind specifically to those sequences in whole cells (Blackburn, 1991; Zakian, 1989). Telomeric DNA sequences and structures are conserved among widely divergent eukaryotes.
  • the essential telomeric DNA consists of a stretch of a G-rich tandemly repeated sequence. Human and other vertebrate telomeres are based on TTAGGG repeat units. The telomere provides a protective "cap" for the end of the chromosome.
  • telomeres prevent loss of genetic information from sub-telomeric regions of the chromosome.
  • Telomeres the ends of eukaryotic chromosomes, are composed of tandemly repeated guanine-rich sequences which have an important role in chromosome organization and stability.
  • the 5' ends of telomeres shorten with each round of replication leaving a 3' overhang that is subject to degradation. This has been described as the "end-replication" problem of linear chromosomes (Watson, 1972; Olovnikov, 1973).
  • the end-replication problem can be overcome by addition of nucleotides to the 3' end of the telomere.
  • a telomere terminal transferase (telomerase) activity was initially discovered in Tetrahymena (Greider & Backburn, 1985).
  • Telomerase activity has since been found in other ciliates (Zahler & Prescott, 1988; Shippen-Lentz & Blackburn, 1989), Xenopus (Mantell & Greider, 1994 ), yeast (Cohn & Blackburn, 1995), mouse (Prowse et al., 1993), and human cells (Morin, 1989).
  • Telomerase is a ribonucleoprotein in which the internal RNA component serves as a template for directing the appropriate telomeric sequences onto the 3' end of a telomeric primer.
  • telomere The cloning (Greider & Blackburn, 1989) and secondary structure determinations of the Tetrahymena telomerase RNA have determined the template portion of the RNA which has suggested a model for the mechanism of telomerase activity.
  • Telomerase is thought to act by: 1) Telomerase binding to the 3' single-stranded overhang of the telomere (TTAGGG in humans) which base pairs with the complementary bases of the RNA component of telomerase, 2) Nucleotide addition onto the 3' end of the telomere by telomerase using its RNA component as a template, and 3) Dissociation of the newly synthesized telomeric DNA from the RNA template and repositioning to allow for the next round of polymerization. This last step is called the translocation step.
  • telomere activity may have an affect on telomerase activity.
  • G-tetraplex structures formed by telomeric sequences may hinder initial telomerase binding (Zahler et ⁇ l, 1991).
  • G-tetraplex formation may actually facilitate the translocation step.
  • G-quartet stuctures may also have a role in telomere function. For example, it has been shown that a variety of proteins will preferentially bind to G-quartet structures (Williamson, 1994). Also, the interaction between guanine-rich DNA strands may be involved in the association of chromosomes seen in cells in the presence of varying concentrations of Na + (Diaz & Lewis, 1975). The function of chromosomal association is unknown but it has been proposed that it is important in such functions as homologous pairing involved in meiosis (Sen & Gilbert, 1988). Recently, a yeast nuclease (Kem1p) was found to specifically recognize and cut only G-quartet structures (Liu & Gilbert, 1994).
  • telomere shortening was shown to cause telomere shortening, cellular senescence, and blockage in the pachytene stage of meiosis in yeast (Bahler et ⁇ l., 1994; Tishkoff et ⁇ l., 1995; Liu et ⁇ l, 1995).
  • telomere length may serve as a "mitotic clock” (Harley, 1995; Shay. 1995). Normal cells in which telomeres shorten to a critical length become senescent (Allsopp et ⁇ l., 1992; Harley, 1991). In contrast, immortal cancer cells have an unlimited replicative capacity.
  • telomere activity is present in a variety of tumor cells, (Chadeneau et ⁇ l., 1995; Counter et ⁇ l., 1994; Counter et ⁇ l., 1995; Kim et ⁇ l., 1994) it appears that activation of telomerase is one link to cellular immortality. This makes inhibition of telomerase an ideal strategy for anti-cancer therapy.
  • a number of nucleoside reverse transcriptase inhibitors show anti-telomerase activity in human and Tetrahymena (Strahl & Blackburn, 1994; Strahl & Blackburn, 1996). 3. Chromosome End Replication Problem
  • Telomeres play a critical role in allowing the end of the linear chromosomal DNA to be replicated completely without the loss of terminal bases at the 5'-end of each strand.
  • Watson (1972) and Olovnikov (1971, 1973) independently described the "end-replication" problem, i.e., the inability of DNA polymerase to replicate fully the ends of a linear DNA molecule.
  • All known DNA polymerases require a primer to initiate polymerization that proceeds in 5' ⁇ 3' direction. After degradation of the RNA primers, filling-in of internal gaps, and ligation events, the parental strand remains incompletely copied.
  • telomerase can be classified as a reverse transcriptase. However, unlike typical reverse transcriptases from retroviruses or lower eukaryotes, it is a ribonucleoprotein that contains its own RNA template as an integral part of the enzyme (Blackburn, 1992). The RNA moiety of telomerase from various ciliates has been cloned and sequenced.
  • the Tetrahymena telomerase RNA moiety is a 159-nucleotide RNA in which a 3'-CAACCCCAA-5' (SEQ ID NO:l) sequence serves as the template for the synthesis of TTGGGG repeats (Greider, 1989).
  • a 15 nucleotide portion, 3'-CAAAACCCCAAAACC-5' (SEQ ID NO:2) of a 191 nucleotide RNA was found that could serve as a template for the synthesis of TTTTGGGG repeats (Shippen-Lentz, 1990).
  • the Euplotes 191 nucleotide RNA and the Tetrahymena 159 nucleotide RNA share little overall primary sequence similarity. However, despite their divergent primary structures, the Euplotes 191-nucleotide RNA, the telomerase RNAs from T. thermophila, and these of other ciliates can all be folded into similar secondary structures with the putative telomeric template domains for each RNA lying in a corresponding position (Shippen-Lentz, 1989). Studies indicate that human telomerase also contains an endogenous RNA as template (Morin, 1989). The RNA component of human telomerase has been cloned (Feng, 1995).
  • telomerase activity from human cells possesses a number of characteristics.
  • TTAGGG G-rich human telomeric primer
  • telomerase activity can be obliterated in the presence of RNase A. It has been shown that the bands formed in the presence of excess cold nucleotides TTP and dATP and limiting amounts of [ ⁇ - 32 P]dGTP (1.56 ⁇ M) are indicative of a pause site at the first guanine in the repeating unit of TTAQGG (Morin, 1989).
  • telomerase is an attractive novel drug target because there is a strong possibility for selectivity.
  • Strahl and Blackburn (1994) has reported that several chain-terminating inhibitors (arabinofuranosyl-guanine triphosphate, Ara-GTP and 2', 3'-dideoxyribofuranosyl guanine triphosphate, ddGTP) efficiently inhibit Tetrahymena and human telomerase (Strahl and Blackburn, 1996). 5.
  • telomeres Early studies on human chromosome ends demonstrated that somatic (peripheral blood) telomeres appeared significantly shorter than germline (sperm) telomeres from the same individual (Cooke, 1986; Allshire, 1988; de Lange, 1990). It is now generally known that in most (if not all) somatic tissues, chromosomes gradually lose their terminal telomere sequence with each cell division (Harley, 1990; Hastie, 1990; Lindsey, 1991; Allsopp, 1992; Shay, 1993; Vaziri, 1993; Klingelhutz, 1994). In contrast, sperm telomeres increase in length with donor age, indicating that telomeres are actively maintained and even elongated in the germline (Allsopp, 1992).
  • telomerase is active in germline cells but somehow is turned off in normal somatic tissues. This hypothesis has some support as no detectable telomerase activity has been found in extracts of embryonic kidney cells, or normal ovarian epithelium (Counter, 1992; Counter, 1994).
  • telomere shortening has an impact on the proliferative activity of somatic cells remains unknown.
  • the minimum telomere length required for maintaining full telomere function has not been fully established.
  • Some evidence suggesting that telomere shortening could play a role in cellular aging comes from the analysis of primary human fibroblasts grown in culture (Harley, 1990; Allsopp, 1992). These cells lose approximately 50 bp per doubling and eventually stop dividing at a senescence stage call Ml. Cells arrested at the Ml stage can be rescued by a variety of viral agents (Counter, 1992; Ide, 1984; Wright, 1989; Radna, 1989).
  • telomere length approximately 1.5 kbp or less as the cell approaches M2 may not contain sufficient telomere sequence to sustain normal telomere function.
  • telomere of the immortal cell lines is stabilized by re-activation of the enzyme telomerase.
  • telomerase activation is an obligatory step in the immortalization of human cells (Counter, 1992).
  • telomerase activity was recently detected in metastatic human ovarian carcinoma cells but not in normal control cells, including healthy ovarian epithelium (Counter, 1994).
  • nucleotide analogs that are incorporated into telomeres by the action of telomerase may interfere with the function of the telomeres.
  • some nucleotide analogs so incorporated may block the ability of telomeres to form G-quarted or G-hairpin structures, thereby rendering the telomeres unable to be recognized by protein which specifically bind these structures.
  • the 3' nucleotides of the overhang region of the chromosome terminus base-pair with a telomere-complementary sequence in the telomerase RNA.
  • the chromosomal end is extended using the RNA as a template, resulting in the addition of six telomeric nucleotides. Then, the extended DNA terminus unpairs from its RNA template and is repositioned on the 3' portion of the template, becoming available for another round of elongation by telomerase.
  • telomerase reaction involves not only copying of an internal template, but also an efficient translocation event which occurs after the last [5' most] residue of the template has been copied into DNA.
  • the translocation step has been deduced from the processive nature of the telomerase reaction in a cell-free assay.
  • telomerase initiates synthesis on a telomeric sequence DNA primer, and in the presence of an excess of the same primer or of a high concentration of a challenging primer, continues to elongate the first primer up to hundreds of nucleotides before dissociation (Blackburn, 1992).
  • telomere preparations are processive in the cell-free assay.
  • Nonprocessive telomerase activity has been described in mouse FM3A cells (Prowse, 1993), Tetrahymena (Collins, 1993) and Xenopus (Mantell, 1994). Both processive and nonprocessive telomerases have been identified by the inventors from different cell lines. Intriguingly, the telomerase in S100 extracts of the human HeLa-S3 subline is nonprocessive, while the telomerase in the parental HeLa cells is processive. Whether these are two different enzymes or the same enzyme with different isoforms is currently being investigated. Whether processivity or non-processivity of the activity identified in the biochemical assay is relevant to telomerase function in whole cells remains to be elucidated. Evidence suggesting that telomerase functions nonprocessively in whole cells has been documented (Blackburn, 1992).
  • telomerase produced in some normal cells there has been no distinction made between the telomerase produced in some normal cells and the telomerase produced by cancer cell.
  • identification of therapeutic compounds which have modulation or inhibitory activity against human telomerase is a desirable goal, particularly to identify compounds and develop methods of treatment of cancers in which processive telomerase contributes to the immortality and undesirable proliferation.
  • telomere activity Toward this end and because of the important if not entirely understood role of telomerase in cell growth and senescence, there has been an effort to identify compounds that affect telomerase activity. Use of such compounds in controlling cell proliferation has obvious implications in treatment of malignant cancers.
  • a goal of current medical investigation is to understand and treat cellular disorders, preferably to selectively target cancer cells either by altering the telomere, the telomerase, or the enzyme structure and/or by inhibiting telomerase.
  • compositions and methods for their use in the inhibition and modulation of eukaryotic telomerase activity More particularly, certain compositions have been shown to modify telomerase activity in cancer cells to more nearly approximate that found in normal or benign cells.
  • telomerase activity in cancer cells to more nearly approximate that found in normal or benign cells.
  • nucleoside triphosphates and their derivatives have been identified as having an inhibitory effect on human telomerase.
  • telomere analogues that inhibit telomerase 7-deaza-2'-deoxyguanosine triphosphate and 7-deaza-2'-deoxyadenosine triphosphate were found to be particularly potent inhibitors of telomerase activity.
  • These compounds were originally investigated based on the rationale that the N-7 nitrogen of purine bases is required for Hoogsteen base-pairing involved in secondary structures formed by telomeric sequences.
  • the 7-deaza nucleotides turned out to be poor substrates for human telomerase and were effective modulators of processive telomerase.
  • the nucleotides thus not only present a novel mode of telomerase inhibition but also are useful for the study of the role of DNA secondary structure in telomerase mechanism.
  • the compounds of the present invention have the general structure (A), (B) or (C)
  • U is independently carbon or nit ogen
  • R 1 is independently H, lower alkyl, or phenyl alkyl
  • R 2 is indepen ently H, lower alkyl, NH 2 , halogen, azido or alkene
  • P is independently oxygen or sulfur
  • S is an acyclic or cyclic glycosyl group represe ⁇ ted by the formulae:
  • R 3 is independen t H, halogen, amino, azido, or hydroxyl; R is independently H or OH; V is oxygen or methylene; and R 5 is OH, (CH 2 ) n PO(OR 7 ) 2 , OP(O)(OR 7 ) 2 ; R 6 is H, lower alkyl, hydroxy-substiruted lower alkyl, or haloalkyl; R is H, lower alkyl, CH 2 OCO(branched or straight chain alkyl (C1 -C8) or aryl, and R 8 is H or CO(lower alkyl) and n is 1-2;
  • K oxygen or methylene
  • G is independently oxygen, sulfur or methyl
  • F is independently oxygen or sulfur
  • the glycosyl bond between S and U is ⁇ or ⁇ .
  • compositions of the present invention found to be useful telomerase-inhibiting analogs may be modified in any of several ways. It is convenient to consider the design of analogs and derivatives in three general categories: (1) modifications of the heterobase of the nucleoside triphosphate. (2) modifications of the ribose sugar; and (3) modifications of the phosphate backbone
  • V O or S
  • R H, Alkyl, Alkene, Alkyne, Amino, Halogen, Azido;
  • R 1 H, Alkyl, pheny
  • the base components of dATP, dGTP, and dTTP are Adenine, Guanine, and Thymine, respectively.
  • the structure of adenine, guanine, and thymine is shown below:
  • adenines such as deaza or azoadenines:
  • modified thymine bases may be used, including: The following general formula summarizes these modifications on the thymine base.
  • V O or S
  • R H, Alkyl, Alkene, Alkyne, Amino, Halogen, Azido;
  • the bond between the base and the sugar moiety may be alpha or beta.
  • the natural nucleoside is the ⁇ -D form.
  • Nonnaturally occurring forms include ⁇ -L, ⁇ -D and ⁇ -L.
  • nucleotide analogs with sugar modification include the 2'- Deoxy-3'-deoxy-3'-substituted nucleosides (or nucleotides):
  • R 1 CH 3 , C 2 H 5 , etc, and Halogen, F, Cl, Br, I etc
  • R 2 H (nucleoside), or any of the modified nucleoside triphosphates
  • R 3 H, F, Cl, NH 2 , N 3
  • sugar moiety need not be limited to any particular sugar and several sugars are contemplated as suitable including in addition to 2'-deoxyribose, ribose, arabinose, xylose, and lyxose
  • Carbocyclic compounds may be substituted for the natural sugars:
  • Acyclic compounds would also be expected to substitute for the sugar residue.
  • R O, S, CH 2 , or OCH 2
  • R 1 H (nucleoside), or any of the modified nucleoside triphosphates
  • the following modified phosphate groups may be attached to appropriate purine or pyrimidine nucleosides, particularly to 7-deaza-2'-deoxyadenosine and 7-deaza-2'-deoxyguanosine residues.
  • Particularly preferred 7-deaza compounds useful for the practice of the present invention are 7-deaza-dGTP and 7-deaza-dATP, having the structures shown:
  • telomerase activity As follows :(i) Both compounds inhibit telomerase in a dose-dependent manner; (ii) 7-deaza-dGTP and 7-deaza-dATP are incorporated into telomeric DNA by telomerase. 7-deaza-dATP can promote non-progressive activity by telomerase. However, incorporation of 7-deaza-dATP or 7-deaza-dGTP results in a telomeric ladder that is prematurely shortened; and (iii) Substrate inhibition (or allosteric inhibition) of 7-deaza-dGTP or 7-deaza-dATP is observed at a high concentration.
  • Q is a protecting group such as a silyl protecting group, e.g. tet-butyl-dimethyl silyl, tetrahydropyranyl, benzyl, etc..
  • a silyl protecting group e.g. tet-butyl-dimethyl silyl, tetrahydropyranyl, benzyl, etc.
  • human telomerase contains a 3'-5' exonuclease activity, analogous to
  • telomere synthesis is a balance of the 3'-5' exonuclease activity and the 5'-3' polymerase activity.
  • synthesis of telomeric DNA can be reduced by blocking the polymerase or by stimulating the exonuclease, or both.
  • Methods for stimulating the exonuclease include altering the interaction of dTTP and possibly other nucleotides with the telomerase. Compounds that block or limit dTTP are contemplated as useful with such a method.
  • Telomerase has a proof-reading-like exonuclease activity, so that incorporated modified nucleotides can be removed with no effect.
  • Such incorporated nucleotides cannot easily be removed by exonuclease activity because the thio-phosphate linkage is not cleaved by exonucleases.
  • Suitable compounds include alpha, beta and gamma thio 7-deaza guanosine, adenosine and thymidine triphosphates such as for example alpha-thio-7-deaza-dGTP.
  • nucleotide analogs appear to also inhibit telomerase polymerization by a competition or allosteric mechanism mediated through telomerase inhibition.
  • telomerase modulators or inhibitors will be of particular use in the treatment of human cancers associated with high levels of processive telomerase.
  • these 2'-deoxynucleosides in order for these 2'-deoxynucleosides to exert their activity, they must be converted to triphosphates intracellularly, i.e., 7-deaza-2'-deoxyguanosine must be transported into the cells and be phosphorylated by a nucleoside kinase to 7-deaza-dGMP and, subsequently, further phosphorylated to 7-deaza-dGDP and 7-deaza-dGTP, respectively.
  • phosphorylation of a nucleoside to its monophosphate is a rate-limiting step in whole cells. If the cells were unable to phosphorylate the nucleosides to monophosphates, no intracellular di- or triphosphates can be formed or identified. On the other hand, one also cannot simply incubate the cells with the phosphates (mono-, di-, or triphosphates) and expect them to transport into the cells and provide the cells with the triphosphates of nucleoside analogs. The phosphates are highly negatively charged, and therefore, will not transport into cells. Instead, the mono-, di-, or triphosphates will be dephosphorylated extracellularly by alkaline phosphorylase or 5'-nucleotidase back to the nucleoside.
  • prodrugs of monophosphates in which the negatively charged phosphate group is functionalized.
  • the prodrug of monophosphates will then contain no charged groups and can be efficiently transported into cells. Once it has entered the cells, the protective group is hydrolyzed by esterase, and nucleoside monophosphate is released. Subsequently, phosphorylation of the liberated monophosphate will produce the desired nucleoside triphosphate.
  • the general structure of the prodrugs is shown below:
  • the present invention has identified one of the most potent telomerase inhibitors
  • 7-deaza-2'-deoxyguanosine-5'-triphosphate The IC 50 value (6.8 ⁇ M) is at least 50-times more potent than AZT-TP. 7-deaza-2'-deoxyguanosine-5'-triphosphate lacks the 7-nitrogen atom which is essential for the formation of G-quartets or hairpins. If 7-deaza-2'-deoxyguanosine-5'-triphosphate is incorporated into telomeres, further telomere elongation may be prevented.
  • -CH r methylene
  • telomere activity 7-deaza-2'-deoxyadenosine-5'-triphosphate (NA023) also inhibits telomerase activity with an IC 50 value of 78.5 ⁇ M); It is now possible to define the structure-activity relationship of nucleoside/nucleotide analogs as inhibitors and modulators of telomerase, thus allowing additional specific inhibitors and modulators of the enzyme to be identified.
  • the design and synthesis of potent telomerase inhibitors based on these studies provide an arrray of telomerase modulating drugs to be used in the treatment of proliferative cell disorders, and particularly those involving cancers characterized by high levels of processive telomerase.
  • telomerase-inhibitory compounds are not believed to be limited in any way to the specific compounds or nucleotide analogs and derivatives specifically disclosed herein. In fact, it may prove to be the case that the most useful pharmacological compounds designed and synthesized in light of this disclosure will be second generation derivatives or further-chemically-modified compositions.
  • telomerase-containing cells are located within an animal
  • a pharmaceutically acceptable composition of the telomerase inhibitor may be administered to the animal in an amount effective to modify the telomerase activity of the target cell.
  • telomere activity In terms of inhibiting telomerase activity in tumor cells, this is contemplated to be an effective mechanism by which to treat cancer that will have very limited side effects.
  • compositions of the present invention find particular utility is the treatment of cell proliferative disorders, and in particular human tumors characterized as having processive telomerase.
  • telomerase inhibitors which either directly inhibit the telomerase activity or indirectly incorporate into telomere and thus prevent telomere further elongation
  • telomere shortening in tumors where telomerase is active. Once the telomere length shortens to a critical length (ca 2 kb), the tumor will go into crisis and eventually die.
  • telomerase inhibitors will have little or no effect on the normal somatic cells because telomerase activity in normal cells is generally low or undetectable.
  • the disclosed compounds will be useful for potentially treating a patient after surgical removal of a tumor.
  • the patient would be treated with non-cytotoxic doses of nucleoside/nucleotide analogs for a prolonged period of time to prevent the recurrence of micro-metastasis.
  • effective treatment of invading pathogens susceptible to telomerase inhibition are also contemplated, as are applications in treating age-related disorders such as atherosclerosis and osteoporosis.
  • telomerase inhibitors there are several methods contemplated by the inventors for the delivery of such telomerase inhibitors into cells.
  • cells are provided with the corresponding nucleoside analogs, and subsequent cellular metabolism converts the nucleosides into nucleoside mono-, di- and tri-phosphates.
  • formulations of specific telomerase inhibitors are prepared in vehicles which protect the nucleotide from phosphatase degradation and facilitate the transport of nucleotides. In the case of the latter, a preferred method for delivery would be that of liposome-mediated delivery.
  • proliferative delivery would be that of liposome-mediated delivery.
  • telomere-mediated transfer of such telomerase inhibitors into human cells is well known to those of skill in the art. Based on existing evidence which shows that the systemic injection of cationic liposome complexes into animals is non-toxic (Stewart et ⁇ l., 1992), the inventors contemplate the use of such liposome-mediated methods for introducing the compositions disclosed herein into animal subjects.
  • Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Chang and Brenner, 1988; Muller et ⁇ l., 1990). Liposomes have been used effectively to introduce drugs (Heath et ⁇ l., 1986; Storm et ⁇ l., 1988; Balazsovits et ⁇ l., 1989), radiotherapeutic agents (Pikul et ⁇ l., 1987), and enzymes (Imaizumi et ⁇ l., 1990; Imaizumi et ⁇ l., 1990) into a variety of cultured cell lines and animals.
  • drugs Heath et ⁇ l., 1986; Storm et ⁇ l., 1988; Balazsovits et ⁇ l., 1989
  • radiotherapeutic agents Pieris et ⁇ l.
  • enzymes Imaizumi et ⁇ l., 1990; I
  • liposome-mediated drug delivery has been completed (Lopez-Berestein et ⁇ l., 1985; Coune, 1988). Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity or gonadal localization after systemic delivery Nabel et ⁇ l., 1992; Mori and Fukatsu, 1992). Introduction of the liposome-telomerase inhibitor complex may be by injection, either systemically into peripheral arteries or veins (including the carotid or jugular vessels), or directly into specific tissues to be targeted.
  • liposome formulations are commercially available, e.g., 1 :1 (w:w) mixture of the cationic lipid n-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) may readily be employed for such liposome formulations.
  • DOTMA n-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOPE dioleoyl phosphatidylethanolamine
  • telomerase activity Based on the inventors' discovery of a method to modify or inhibit telomerase activity, it is contemplated that several classes of compounds will be useful. It will be desirable to determine which analogs and derivatives will be most suitable for particular treatments; for example, depending on the type of cancer cell present and particularly the amount and activity of telomerase present.
  • a related aspect of the invention is the discovery that certain allosteric interactive agents will alter or inhibit telomerase activity. Nucleotides such as 7-deaza dGTP and 7-deaza dATP, are capable of allosterically inducing a reduction in telomerase polymerizing activity. The inventors have demonstrated that other nucleotides such as dGTP will not induce this effect, indicating the importance of the 7-deaza modification.
  • telomere This now provides a new method of modulating telomerase for the treatment of cancer by designing a series of 7-deaza compounds that will allosterically bind with telomerase with varying effects on modulating telomerase activity.
  • Highly processive telomerase for example may require stronger inhibitors or modifications to the nucleotide analog that change allosteric interactions or are less efficiently incorporated into the telomere.
  • FIG. 1 Telomerase activity from HeLa (processive) versus UA 21 (non-processive) cells. RNase prevents telomerase activity.
  • FIG. 3 Inhibition of telomerase ladder with 7-deaza-dGTP (A) or dGTP (B) in the presence of 1.56 ⁇ M [ ⁇ - 32 P]dGTP containing 1 mM dTTP, 1 mM dATP, 1 ⁇ M
  • TTAGGG 3 , 20 ⁇ l S100, and from left to right: 0 (C), 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100 ⁇ M 7-deaza-dGTP or dGTP.
  • telomerase ladder with 7-deaza-dATP (C) or dATP (D) in the presence of 3.12 ⁇ M [ ⁇ - 32 P]dATP. containing 1 mM dTTP, 1 mM dGTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l S100, and from left to right: 0 (C), 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100 ⁇ M 7-deaza-dATP or dATP.
  • FIG. 4. (A) Inhibition of telomerase ladder with 7-deaza-dGTP in the presence of 3.12 ⁇ M [ ⁇ - 32 P]dATP. containing 1 mM dTTP, 1 mM dGTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l SI 00, and from left to right: 0 (C), 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0 mM 7-deaza-dGTP
  • (B) Inhibition of telomerase ladder with 7-deaza-dATP in the presence of 1.56 ⁇ M [ ⁇ - 32 P]dGTP containing 1 mM dTTP, 1 mM dATP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l SI 00, and from left to right: 0 (C), 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0 mM 7-deaza-dATP.
  • FIG. 5 A 7-deaza-dGTP as a telomerase substrate with 3.12 ⁇ M [ ⁇ - 32 P]dATP, 1 mM dTTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l SI 00 from left to right: 1 mM dGTP (C), and 0, 0.25, 0. 5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0 mM 7-deaza-dGTP.
  • the arrows point to the triplet band most prominent at 0.75 and 1.0 mM 7-deaza-dGTP.
  • the arrows point out the triplet bands.
  • FIG. 5C dGTP as a telomerase substrate with 3.12 ⁇ M [ ⁇ - 32 P]dATP, 1 mM dTTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l S100 from left to right: 1 mM dGTP (C), and 0, 0.25, 0. 5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0 mM dGTP.
  • FIG. 6A 7-deaza-dATP as a telomerase substrate with 1.56 ⁇ M [ ⁇ - 32 P]dGTP, 1 mM dTTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l S100 from left to right: 1 mM dATP (C), and 0, 0.25, 0. 5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0 mM 7-deaza-dATP.
  • FIG. 6B 7-deaza-dATP as a telomerase substrate with 1.56 ⁇ M [ ⁇ - 32 P]dGTP, 1 mM dTTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l S100, 1 mM 7-deaza-dATP without (-) or with (+) RNase A at 0.125 ⁇ g/ ⁇ l.
  • FIG. 6C dATP as a telomerase substrate with 1.56 ⁇ M [ ⁇ - 32 P]dGTP, 1 mM dTTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l S100 from left to right: 1 mM dATP (C), and 0, 0.25, 0. 5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0 mM
  • FIG. 7 Telomerase activity with 1 mM dTTP, 1 ⁇ M (TTAGGG) 3 , 20 ⁇ l S100, 1.56 ⁇ M [ ⁇ - 32 P]dGTP 1 mM dATP (dGTP*) or 3.12 ⁇ M [ ⁇ - 32 P]dATP and 1 mM dGTP (dATP*). Reactions contained either no RNase A (-) or 0.125 ⁇ g/ ⁇ l of RNase A (+).
  • FIG. 8 Temperature dependence of the 1H NMR spectrum of the human telomeric DNA sequence d(GGTTAGGGTTAG) in NaCl- and Kcl-containing buffers.
  • FIG. 9 Temperature dependence of the ⁇ NMR spectrum of the human telomeric DNA sequence d(GGTTAGG*GTTAG) where G* is 7-deaza-2'-deoxy guanosine.
  • FIG. 10 Incorporation of NA004 by Human Cancer Telomerase.
  • FIG. 11 Incorporation of NA006 by Human Cancer Telomerase.
  • FIG. 12 Lack of Incorporation of NA007 by Human Cancer Telomerase.
  • FIG. 13 Incorporation of NA013 by Human Cancer Telomerase.
  • FIG. 14 Incorporation of NA014 by Human Cancer Telomerase.
  • FIG. 15 Lack of Incorporation of NA020 by Human Cancer Telomerase.
  • the present invention provides several nucleoside triphosphates which are capable of inhibiting mammalian telomerase activity.
  • the compounds show differential inhibition against telomerase in S100 extracts of transformed human fetal kidney tumor 293 cells (a processive telomerase) versus that of hamster UA21 cells (non-processive telomerase). This observation has prompted the investigation of nucleoside/nucleotide analogs which are useful in affecting telomere/telomerase function in cells.
  • telomeres While inhibition of telomerase represents one way of interfering with the function of cellular telomeres, providing DNA reactive drugs which damage telomeric DNA itself is a possible complementary approach. Damage to telomeres is more c.3trimental to rapidly growing (i.e. tumor) cells than to normal cells. Given the significance of telomeres, compromised telomere integrity results in incomplete replication of telomeric DNA, disturbed chromatin structure and, eventually, cell death. An attempt to replicate telomeres with unrepaired lesions may further amplify the initial damage. Thus, tumor cells that have very short telomeres would be the most susceptible to incomplete telomere replication. Moreover, lesions in telomeric DNA may be more lethal in those tumor cells that require telomerase to actively maintain their already shortened telomeres.
  • telomeres are highly repetitive sequences (TTAGGG) n it is possible that DNA-reactive agents with a preference for A/T or G/C may damage telomeric DNA to a different extent than other regions of the chromosome. Also, the repair of damage in telomeres is likely to be slower than repair in the rest of the genome (Kruk and Bohr, 1993).
  • telomeres may affect telomeres in addition to inducing damage to other regions of the genome. Lesions in telomeres may disturb telomerase action. For example, bifunctional alkylating agents may form crosslinks between telomeric DNA and telomerase protein and/or RNA components. Such adducts will probably abrogate enzyme activity. Conversely, inhibition of telomerase may enhance lethality of drug-induced DNA lesions in other regions.
  • telomere maintenance allows cancer cells to be immortal without the danr _r of chromosome instability. While normal cells do not maintain or grow their telomt res nor cell do produce a regulated and non-aggressive form of telomerase characteri. ;d by its non-processive activity. This activity appears to be necessary for certain fun tions in normal cells other than maintaining or growing telomeres.
  • telomere Unlike cancer cells, normal cells are not immortal, ostensibly due to lack of or very low levels of proces. ive telomerase. This information indicates that a method for converting processive teiomerase to non-processive telomerase in a way that would not harm normal cells but w >uld selectively affect cancer cells.
  • the present invention den- onstrates a method of altering processive telomerase so that it acts non-processively. T his modulation causes cancer cell telomerase to more closely mimic normal cell non-processive telomerase.
  • changes to convert processive telomerase to non-processive telomerase may involve either direct changes to the telomerase complex itself or indirect methods that ultimately affect telomerase activity. Direct methods for example may involve cleaving the RNA component into partial fragments, such that only processive activity is affected.
  • Processive telomerase activity may be modulated to nonprocessive activity by a partial digest treatment with RNase A or with other RNA cleaving agents, such as ribozymes. Similar results may be obtained by chemical modification of the RNA, or binding of agents, such as oligonucleotides.
  • the present invention illustrates an indirect method for converting processive telomerase to nonprocessive telomerase by allowing telomerase to incorporate 7-deaza-dGTP or related analogs into telomeric DNA.
  • 7-deaza-dGTP and its analogs once incorporated into the telomere, alter the telomeric secondary structure so that telomerase can no longer recognize it properly for processive telomere synthesis.
  • telomere analogs can be incorporated in the telomere by any polymerase capable of replicating the telomere. Once in place, only the telomeric sequence is affected since the unique G-rich repeat structure of telomeric DNA is found only in the telomere.
  • the inventors' strategy is to provide selected nucleoside analogs that act in two ways; both to alter processive telomerase to act non-processively and to affect telomere structure so that telomere-binding proteins will no longer recognize the telomere. The telomere is thus left unprotected, resulting in chromosome instability and cell death. Cancer cells will be selectively targeted because these cells depend on processive telomerase to incorporate the nucleotide analog. The more telomerase that a cell produces, the more effective the strategy. The method also has advantages over merely inhibiting telomerase, because the telomere is immediately affected and it is not necessary to wait for the telomere to shorten and become dysfunctional.
  • the disclosed method of using selected nucleoside analogs to alter telomerase activity does not inhibit telomerase but merely converts one form of telomerase to another form of telomerase.
  • telomere activity has been identified in the following cell lines: 293 (a transformed human embryonic kidney cell line), HeLa and HeLa-S3 (a human cervical tumor cell line), CEM (a human leukemia cell line) and UA-21 (a subline of Chinese hamster cell CHO line).
  • processive telomerase activity has been identified in the S100 extracts of 293, CEM, HeLa cell lines, and non-processive telomerase activity in the S100 extracts of HeLa-S3, UA-21 and WI38 cell lines (FIG. 1).
  • telomeres were tested in one or both of the "conventional" or “modified” telomerase assays described in Methods using the human processive and the CHO non-processive telomerases. Initially, three chain-terminators were selected: 2', 3'-dideoxythymidine triphosphate (ddTTP), 3'-fluoro-2',3'-dideoxythymidine triphosphate (F-ddTTP), and 3'-azido-2-', 3'-dideoxythymidine triphosphate (AZ-ddTTP) as prototype agents to investigate whether they could inhibit telomerase activity.
  • ddTTP 3'-dideoxythymidine triphosphate
  • F-ddTTP 3'-fluoro-2',3'-dideoxythymidine triphosphate
  • AZ-ddTTP 3'-azido-2-', 3'-dideoxythymidine triphosphate
  • the three compounds are dTTP analogs without a 3'-OH group for subsequent chain elongation. They may either incorporate into the telomere sequence and terminate further chain elongation or compete directly with dTTP for the substrate binding site in the telomerase reaction. In either case, telomerase activity is inhibited.
  • Results showed that AZ-ddTTP inhibits both the human processive and the CHO non-processive telomerase in a dose-dependent manner.
  • the DNA ladder bands were quantitated using a Molecular Dynamic Personal Densitometer.
  • the concentration of AZ-ddTTP required to inhibit the telomerase activity by 50% of the control is estimated to be 500 and 750 ⁇ M against the 293 and UA-21 telomerase, respectively.
  • the nucleoside AZT (3'-azidothymidine), at 2 mM, has a small effect on the telomerase activity.
  • nucleoside triphosphate is an inhibitor of telomerase.
  • Inhibition studies are typically performed at concentrations of dTTP and dATP (2 mM) which could be much higher than the K m of dTTP and dATP for telomerase.
  • the inventors tried lower concentrations of dTTP and dATP (near the K m ; in the ⁇ M ranges) and showed that AZ-ddTTP is actually a more potent inhibitor of telomerase than previously estimated. It was found that ladder patterns were detectable even at dTTP and dATP concentrations as low as ⁇ 200 ⁇ M.
  • dTTP analogs were studied. A compound with a 3 '-amino substitution had only a marginal effect on the human telomerase activity. However, another compound, 4-thiothymidine triphosphate, with a 4-thio group rather than a 4-keto group on dTTP, inhibited the human processive enzyme completely at 1 mM. 4-thiothymidine triphosphate has a 3'-OH group and, therefore, appears not to chain-terminate the telomerase reaction when incorporated into the nascent DNA strand. This is the first observation that a dNTP analog with a heterocyclic ring modification has an effect on telomerase activity.
  • ddATP 2'3'-dideoxyadenosine triphosphate
  • ddTTP 2'3'-dideoxythymidine triphosphate
  • the new analogs have the following structure.
  • AZ-ddTTP is a negatively charged molecule and cannot be transported into cells.
  • CHO cells were incubated with 800 ⁇ M (non-cytotoxic concentration) of 3'-azidothymidine (AZT nucleoside) for several generations.
  • the effect of AZT on the telomere length and on the formation of unstable dicentric chromosomes was determined as an indirect measurement of the effect of AZ-ddTTP on intracellular telomerase activity.
  • An initial, gradual shortening of the telomere length was found with a concurrent increase in the number of dicentric chromosomes in the AZT-treated CHO cells. This effect was only transient; after 20 generations, the fraction of cells with dicentric chromosome decreased.
  • AZddTTP inhibits both the 293 and UA-21 enzymes.
  • the frequency of dicentric chromosomes increased in the presence of AZT. Eventually, the cells stopped growing and apparently died.
  • AZ-ddTTP AZT triphosphate
  • AZT triphosphate AZT triphosphate
  • telomerase activity studies were initiated to determine intracellular AZT triphosphate concentrations.
  • the metabolism of AZT is species-and cell type-specific (Balzarini, 1988; Balzarini 1989).
  • CHO cells using 3 H-labeled AZT, it was found that the uptake (transport and subsequent metabolism to its phosphate metabolites) of AZT was concentration- and time-dependent.
  • AZT accumulates intracellularly at concentrations 2-3 times higher than the extracellular concentrations (0.25-750 ⁇ M) tested. Total AZT uptake reached a plateau in approximately 30 minutes.
  • FIGS. 3A and 3C illustrate the effect of 7-deaza-dGTP and 7-deaza-dATP on the level of telomerase activity.
  • Inhibition of telomerase activity was indicated by a reduction in the intensity of the bands with increasing concentrations of inhibitor.
  • the degree of inhibition was determined by measuring the intensity of the bands in the telomerase ladder with densitometry.
  • the intensities in each lane were normalized to an internal standard, 32 P 5' end-labeled (TTAGGG) 3 , to compensate for differences in processing of the products and gel loading. Intensities in all lanes were exr.essed as a percent of the control to eliminate variability due to differences in film jxposure and enzyme preparations.
  • IC 50 concentration of inhibitor that results in a 50% reduction in telomerase activity
  • 7-Deaza-dGTP and 7-deaza-dATP can also inhibit telomerase activity even in excess (1 mM) dGTP or dATP respectively (FIG. 4A and FIG. 4B).
  • the IC 50 value of 7- deaza-dGTP as a telomerse inhibitor in the presence of 1 mM dGTP, dTTP and 3.12 ⁇ M [ ⁇ - 32 P]dATP was 56 ⁇ M.
  • the IC 50 of 7-deaza-dATP was 59 ⁇ M when in the presence of 1 mM dATP, TTP and 1.56 ⁇ M [ ⁇ - 32 P]dGTP.
  • I. 7-Deaza-dGTP and 7-deaza-dATP are telomerase substrates
  • Telomerase paused at significantly more sites in the presence of 7-deaza-dGTP.
  • 7-deaza-dGTP At a concentration of 0.5 mM 7-deaza-dGTP, there was such a large number of bands pertaining to various pause sites that a predominant pause site could not be determined.
  • 7-deaza-dGTP concentration 0.75-1 mM, a repeating triplet of pause sites was distinguishable (FIG. 4A lanes 5 and 6, FIG. 5B). These bands appeared to be two, three, and four bases smaller than that with dGTP (FIG. 5A control lane) corresponding to the guanines in the TTAGGG repeat.
  • telomere ladder was difficult to obtain with concentrations less than 3.12 ⁇ M of [ ⁇ - 32 P]dATP.
  • a telomerase ladder was not generated in the presence of limiting radioactive dTTP.
  • the compounds 7-deaza-dGTP and 7-deaza-dATP have been found to be potent inhibitors and modulators of telomerase activity in the S100 extracts of 293 cells.
  • the level of inhibition of the radioactive native substrate by these compounds is comparable to that caused by the cold native substrate.
  • the 7-deaza nucleotides are able to inhibit telomerase activity with IC 50 values of less than 100 ⁇ M.
  • 7-Deaza-dGTP and 7-deaza-dATP can replace the native purine nucleotides, dGTP and dATP respectively.
  • the total activity is weak and shorter ladders are formed when the 7-deaza analogues replace their native nucleotides.
  • 7-deaza purine nucleotides are effective substrates for a variety of other DNA polymerases (Mizusawa et ⁇ l., 1986; McConlogue et ⁇ l., 1988; Seela & Roling, 1991).
  • N7 is a factor in telomerase processive activity.
  • N7 is involved in G-quartet and hairpin structures. Replacing dG with di in various telomeric sequences disrupted G-tetraplex formation but had no inhibitory effect on telomerase in vitro (Henderson et ⁇ l., 1990). Furthermore, it has been shown that G-quartet structures may actually inhibit the initiation of telomerase activity (possibly by prevention of binding of telomerase to the oligonucleotide). However, it has been proposed that G-tetraplex structures can facilitate the translocation step (Zahler et ⁇ l., 1991). Incorporation of the 7-deaza nucleotides can prevent formation of these structures.
  • the oligodeoxyribonucleotide having the sequence d(GGTTAGG*GTTAG) when G* represents 2'-deoxy-7-deazaguanosine, in KCL-containing buffer displays only weak and broad imino proton signals in the G-quartet dianostic region of 10-12 ppm in its low temperature 1H NMR spectrum. These weak signals disappear as the sample is warmed, and are no longer present at 25° C (FIG. 9) making the translocation step more difficult. The result of this would be formation of shorter telomerase products.
  • the telomerase ladder formed in the presence of 7-deaza-dATP is also prematurely shortened.
  • alternating d(A-T) oligomers in which dA is replaced by 7-deaza-dA are slightly more stable than d(A-T) oligomers.
  • Replacing dA with 7-deaza-dA in homoligomers dAdT also results in a decrease in stability.
  • alternating d(7-deazaA-T) oligomers are slightly more stable than d(A-T) oligomers (Seela & Thomas, 1995).
  • replacing dA with 7-deaza-dA within poly dA tracts reduces the degree of bending (Seela et ⁇ l., 1989).
  • Dissociation of the enzyme from the growing strand may account for decrease in processivity of telomerase as the 7-deaza nucleotides are incorporated. Furthermore, a change in stability or conformational change caused by addition of one or more 7-deaza-dG's per TTAGGG repeat may cause telomerase to dissociate during the elongation step. This could be the cause of the shift and increase in number of pause sites observed in the presence of 7-deaza-dGTP but not 7-deaza-dATP.
  • a third explanation involves substrate inhibition.
  • the substrate inhibition seen at higher 7-deaza-dGTP and 7-deaza-dATP concentrations has been observed for a variety of other enzymes (Dixon & Webb, 1979). Substrate inhibition is also seen in
  • Substrate inhibition could be due to two mechanisms (Dixon & Webb, 1979).
  • the nucleotides can bind to the telomerase active site to form other ineffective telomerase-nucleotide complexes which inhibit telomerase activity.
  • the less effective binding modes have a higher K m than the correct mode so that inhibition becomes more apparent only when higher concentrations of substrate is present.
  • the nucleotides can bind to a site other than the active site which causes a conformational change resulting in inhibition. This binding would have a lower affinity than the active site binding so that inhibition becomes more apparent with increasing substrate concentrations.
  • the 7-deaza nucleotide competes with the radioactive nucleotide at limiting concentrations, 1-3 ⁇ M, the observed IC 50 value most likely reflects the direct competition between the cold and labeled nucleotide.
  • the observed inhibition of telomerase activity by the 7-deaza nucleotides in the presence excess concentrations of the nonradioactive native nucleotide may be due to substrate inhibition. Details of human telomerase structure are unknown at this time; thus, how the substitution of a carbon for N7 is responsible for the observed substrate inhibition is not yet fully defined.
  • compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit.
  • the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavor
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus ny additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the compounds may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • aqueous composition that contains an active ingredient is well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • composition can be formulated in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Tris-HCl Tris(hydroxymethyl)-aminomentane hydrochloride
  • MEM minimal essential medium
  • HEPES N-[2-Hydroxyethyl]piperazine-N'-[2-ethansulfonic acid]
  • PMSF Phenylmethylsulfonic acid
  • EDTA ethylenediaminetetraacetate
  • dGTP 2'-deoxyguanosine-5'-triphosphate
  • dATP 2'-deoxyadenosine-5'-triphosphate
  • TTP 2'-deoxythymidine-5'-triphosphate
  • dG 2'-deoxyguanosine
  • dA 2'-deoxyadenosine
  • dC 2'-deoxycytidine
  • Oligonucleotide primers (Genosys), dNTP's including 7-deaza-dGTP and 7-deaza-dATP (Pharmacia), [ ⁇ - 32 P]dGTP and [ ⁇ - 32 P]dATP (Dupont NEN), RNase A, PMSF, pepstatin A, and leupeptin, and other chemicals (Sigma).
  • Telomerase was prepared from transformed human embryonic kidney 293 cell line.
  • Human embryonic kidney 293 cells were grown in Joklik's modified MEM medium (Sigma, St Louis, MO) supplemented with 5% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2- The cells were subcultured at 1-2 ⁇ 10 5 cells/ml twice a week.
  • the cells When the cells grew to approximately 1 x 10 ⁇ cells/ml, the cells ( ⁇ 2 ⁇ 10 9 cells) were centrifuged and transferred into polypropylene centrifuge (50 ml) tubes and kept on ice. The cells were then centrifuged in a (Beckman GS-6R) centrifuge at 1200 RPM for 10 minutes at 4°C, and resuspended in PBS.
  • the washed cells were resuspended in 15 ml Hypobuffer Mix (10 mM HEPES, pH 8.0, 3 mM K Cl, 1 mM MgCL 2 , 1 mM dithiothreitol, 1 ⁇ M leupeptin, 0.1 mM PMSF, 10 ⁇ M pepstatin, 0.83 ⁇ g/ml chymostatin, 0.83 ⁇ g/ml antipain, and 43.2 units/ml RNase Guard) and centrifuged. The cell pellets were resuspended in 0.75 ⁇ volumes Hypobuffer [Hypobuffer Mix + 40 units/ml RNase Guard (Pharmacia, NJ) to allow the cells to swell.
  • Hypobuffer Mix 10 mM HEPES, pH 8.0, 3 mM K Cl, 1 mM MgCL 2 , 1 mM dithiothreitol, 1 ⁇ M leupeptin, 0.1 mM PMSF, 10 ⁇
  • the cell suspensions were homogenized to allow the telomerase to "leak" into the cytosolic fraction.
  • the lysate was then poured into new, autoclaved, 3 ml polyallomer ultracentrifuge tubes and centrifuged at 100,000 x g for 1 hour at 4°C. A top lipid layer was carefully removed and discarded, and the supernatant was collected. 1/50 of the volume of 5M NaCl was added to the clear supernatant fluid to achieve a final concentration of 0.1 M. Sterile glycerol was added until the final glycerol concentration was 20%.
  • the enzyme solution was aliquoted into Eppendorf tubes and quickly frozen in liquid nitrogen and stored at -70°C.
  • the assay is based on the ability of the telomerase enzyme in the S100 fractions to recognize an 18-base DNA primer with the DNA sequence (TTAGGG)3.
  • DNA polymerization initiates from the 3'-OH group of the oligonucleotide.
  • telomerase adds a repeated set of 5'- TTAGGG-3' to the original 18-base primer.
  • the telomerase assay was initiated by mixing 20 ⁇ l of 2x reaction mix (100 mM Tris-OAc, pH 8.5, 100 mM KOAc, 10 mM ⁇ -mercaptoethanol, 2 mM spermidine, 2 mM MgCl 2 , 2 mM dATP, 2 mM dTTP, 2 ⁇ M oligo primer, potential inhibitors of interest at the appropriate concentration, and 50 ⁇ Ci [ ⁇ 32- P]dGTP) with 20 ⁇ l of S100 enzyme extract. The reaction mixture was then incubated at 30°C for 1 hour.
  • 2x reaction mix 100 mM Tris-OAc, pH 8.5, 100 mM KOAc, 10 mM ⁇ -mercaptoethanol, 2 mM spermidine, 2 mM MgCl 2 , 2 mM dATP, 2 mM dTTP, 2 ⁇ M oligo primer, potential inhibitors of interest at the appropriate concentration, and 50 ⁇
  • the reaction was stopped by incubating sequentially with 50 ⁇ l of the stop solution (10 mM Tris pH 7.5, 20 mM EDTA, 0.1 mg/ml RNase A) and 50 ⁇ l of deproteinase solution (10 mM Tris pH 7.5, 0.5% SDS, 0.3 mg/ml Proteinase K). The mixture was extracted with 50% phenol/50% chloroform (v/v). The DNA was then precipitated by adding 2.5x volumes of cold EtOH and letting it stand overnight at -70°C . The DNAs were separated on an 8% polyacrylamide/7M urea denaturing gel at 1600 V for 1.5 hours. The gel was dried and exposed to Kodak XAR-5 film to gel in -70°C . Typical autoradiographic exposures required 3-14 days. 3. Modified Telomerase Assay ( 32 P-d ATP as Radiolabeled Substrate)
  • the concentrations of both non-radiolabeled nucleotides, dATP and dTTP are 1 mM, and the concentration of the radiolabeled
  • [32p]dGTP is 1.56 ⁇ M. Because of the high concentration of dATP and dTTP, the standard assay may not be suitable for determining the potential telomerase inhibition of any "Adenine or Thymine" analogs.
  • a modified alternative telomerase assay was developed which uses dGTP (1 mM) and dTTP (1 mM) as the non-radiolabeled substrates and 3.12 ⁇ M [ ⁇ 32 P]-dATP as the radiolabeled substrate. All other chemicals and reaction conditions are identical to the conventional assay. 4. Testing Selected Nucleoside Triphosphates as Telomerase Substrates
  • telomerase catalyzes the incorporation of dATP, dTTP and [ ⁇ - 32 P]dGTP into a (TTAGGG) 3 primer.
  • native dATP in the standard telomerase reaction is replaced with various concentrations of dATP analogs.
  • telomeric products are isolated and analyzed as described in Section 2.
  • native dTTP in the standard telomerase reaction can be replaced by a structurally modified dTTP analog to test whether the dTTP analog can be used as a telomerase substrate.
  • dGTP analog In order to evaluate whether or not a dGTP analog can be incorporated by telomerase, native dGTP in the modified telomerase reaction (1 mM dGTP, 1 mM dTTP and 3.12 ⁇ M [ ⁇ 32 P]dATP) is replaced with the dGTP analog.
  • the oligodeoxyribonucleotide having the sequence d(GGTTAGGGTTAG), corresponding to the human telomeric DNA sequence was prepared using standard automated solid phase synthesis techniques employing a ten micromole synthesis on an Applied Biosystems Model 381 A automated DNA synthesizer. Solid support and reagents were purchased from Biogenex, Glen Research, or Applied Biosystems, South San Francisco, CA. The DNA sample was deprotected in concentrated NH 4 OH at 55°C overnight, purified by reverse phase HPLC on a C18 column (Dynamax-300A) and then dialyzed extensively against deionized water.
  • NMR samples were prepared by reconstituting lyophilized DNA in 90% H 2 O/10% D 2 O containing 1 mM EDTA/50 mM potassium phosphate and either 100 mM KCl or 100 mM NaCl. NMR experiments were carried out on a Bruker AMX 500 MHz spectrophotometer using a 1-1 echo pulse sequence with maximum excitation centered as 12.0 ppm. At temperatures as high at 70°C, the NMR spectrum of the sample disolved in KCl buffer displayed characteristic resonances in the region of 10-12 ppm that corresponded to the imino protons of the G-quartet structure. The same sample dissolved in NaCl buffer only displayed these imino resonances at 10-12 ppm up to temperatures of 60°C, indicating that these resonances correspond to the potassium-stabilized G-quartet structure.
  • the fully protected phosphoramidite of a selected nucleotide analog was used in an automated solid phase DNA synthesizer to synthesize a oligodeoxyribonucleotide having the same sequence but with one natural base substituted with the nucleotide analog, for example d(GGTTAGG*GTTAG) where G* represents a 7-deaza-2'-deoxyguanosine residue.
  • the oligodeoxyribonucleotide is dissolved in 90% H 2 O/10% D 2 O containing 100 mM KCl/1 mM EDTA/50 mM potassium phosphate.
  • Exponentially growing cells (1-2 ⁇ 10 3 cells, unless specified otherwise) in 0.1 ml medium were seeded on day 0 in a 96-well microtiter plate.
  • 0.1 ml aliquots of medium containing graded concentrations of test analogs were added in duplicate to the cell plates. After incubation at 37°C in a humidified incubator for 6 days, the plates were centrifuged briefly and 100 ⁇ l of the growth medium was removed.
  • a m ⁇ x is the absorbance of control cells
  • a min is the absorbance of cells in the presence of highest agent concentration
  • Y is the observed absorbance
  • X is the agent concentration
  • IC 50 is the concentration of agent that inhibits the cell growth by 50% of control cells (based on the absorbance)
  • n is the slope of the curve.
  • Tumor cells were incubatec with graded concentrations of [ 3 H]AZT at 37°C. At the indicated intervals, 2 ⁇ 10 7 cells were centrifuged at 1000 x g for 6 min and were washed once with 5 ml of cold 0.9% NaCl. Cold perchloric acid (4%; 0.45 ml) was added to the pellets, and the tubes were vortexed vigorously. The cold perchloric acid mixture was placed in ice for 20 min and was vortexed occasionally. The precipitated protein and nucleic acids were removed by centrifugation at 3000 x g for 20 min at 4°C.
  • HPLC analysis was performed on a Beckman HPLC. The separation was achieved by a linear gradient elution of potassium phosphate buffer pH 4.5 (10 to 500 mM) on a Whatman strong anion exchange column, Partisil 10 SAX.
  • HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum in the presence of testing agents (at the indicated concentration ⁇ IC10 value). The cells were subcultured every 3.5 days. The cells were grown for 4 passages which is equivalent to ⁇ 12 cell population doublings. At the appropriate cell passage, ⁇ 1 ⁇ 10 7 HeLa cells were harvested by trypsin treatment. High quality DNA (large in size) was prepared by Proteinase K and RNase treatment, followed by phenol extraction. DNA isolated from the control and analog-treated HeLa cells was digested with restriction enzyme MseI to completion. Restriction digested DNAs were electrophoresed on a 0.8% agarose gel.
  • the gel was Southern blotted onto a nitrocellulose or nylon membrane and the membrane was probed by hybridization with a telomere specific sequence containing TTAGGG repeats.
  • the membrane was exposed to autoradiographic film. After development, the signals in each lane were scanned and quantitated by video (CCD) densitometry. The median length, peak, and other curve characteristics were determined by computer (using Image 1.5). Duplicate samples were analyzed for each compound. 9. Induction of Dicentric Chromosome by aTelomerase Inhibitor
  • telomere The same cells grown for the telomere analysis were plated for chromosome preparation. Metaphase cells were harvested, fixed by methanol/ acetic acid and the chromosomes spread on slides for fluorescent in situ hybridization analysis. The spreads were hybridized with a centromere probe and detected by a fluorescein tag. The number of fused chromosomes per cell was calculated for each compound tested.
  • Telomerase was prepared from various human cancer cells and normal stem cells. Telomerase activity was compared using the conventional telomerase assay (see section 2 under Materials and Methods) and modified conventional telomerase assay see section 3 under Materials and Methods). Telomerase incorporates labeled dGTP into a telomeric primer by adding a telomeric repeat or repeats. The labeled primers were electrophoresed and visualized by autoradiography. Telomerase activity is indicated below in the first lane on the left is from human renal cell carcinoma cancer cells. The telomerase product bands are indicated by numbered arrows. This activity is processive as indicated by the multiple bands produced by the cancer telomerase adding multiple telomeric repeats. The telomerase activities in lane marked 1,2, and 3 are from normal human blood stem cells. These normal telomerase activities are non-processive as indicated by the single band produced by normal telomerase adding a single repeat to the primer.
  • telomerase Human cancer telomerase was incubated with limited amounts of RNase A, and then assayed using the conventional telomerase assay where labeled dGTP is incorporated into the telomerase products. The products were separated by electrophoresis, and visualized by autoradiography. The left lane of autoradiogram below shows the human cancer telomerase activity without treatment and the right lane shows the same telomerase activity with RNase treatment. The limited amount of RNase partially cleaved the telomerase RNA component so that the telomerase acts non-processively. This is consistent with the RNA component having structures required for the processive function of telomerase. These structures can be targeted to convert a cancer's aggressive processive telomerase activity to a non-aggressive or benign and non-processive activity.
  • Human processive telomerase exhibits a 3' to 5' exonuclease activity that can be stimulated to remove telomeric sequence rather than synthesize telomerase sequence.
  • Human cancer telomerase was incubated in the presence of a limited amount of dTTP.
  • the autoradiogram below shows the exonuclease activity of telomerase where a reduction in nucleotides such as dTTP and a change in the DNA primer substrate can induce the exonuclease activity.
  • the exonuclease removes G residues from the primer, the polymerase of telomerase can fill them back in, thus labeling the original primer with labeled dGTP.
  • Telomerase activity was determined by the conventional assay using an 18-mer telomeric sequence primer, and the products were separated by electrophoresis. The two left lanes show products revealing that telomerase has removed a significant amount of the telomeric sequence as the result of a 3' to 5' exonuclease. This activity was stimulated by the limitation of dTTP (e.g. 100mm) and is not normally observed under normal conditions (e.g. lmm).
  • telomeres with modifications on the 2'-deoxyribose moiety or on the heterocyclic moiety are capable of inhibiting human telomerase activity.
  • the studies were performed using the conventional telomerase assay in which the competing dTTP concentration is in the range of 1-2 mM.
  • Telomerase was prepared from 293 cells. The conventional .elomerase assay was performed where telomerase incorporates labeled dGTP into a fr lomeric primer and the labeled primers are electrophoresed and signal quantitated by autoradiography and densitometry. Telomerase was incubated with concentrations of AZTTP ranging from 0 to 2 mM. The IC 50 was ⁇ 200 ⁇ M. The relative percent activity of AZTTP at different concentrations is shown below.
  • Telomerase was prepared from 293 cells. The conventional telomerase assay was performed where telomerase incorporates labeled dGTP into a telomeric primer and the labeled primers are electrophoresed and signal quantitated by autoradiography and densitometry. Telomerase was incubated with concentrations of ddTTP ranging from 0 to 2 mM. The IC 50 was ⁇ 500 ⁇ M. The relative percent activity of the ddTTP with increasing concentration is shown below.
  • Telomerase was prepared from 293 cells. The conventional telomerase assay was performed where telomerase incorporates labeled dGTP into a telomeric primer and the labeled primers were electrophoresed and signal quantitated by autoradiography and densitometry. Relative activity change with concentration is shown in the graph below. Telomerase was incubated with concentrations of 3'-Amino-TTP ranging from 0 to 2 mM. The IC 50 was -2 mM.
  • NA021 is an analog of dTTP in which the methyl group is replaced with an allylamino group.
  • NA021 demonstrated a concentration dependent inhibition of 293 cell telomerase with an estimated IC50 value of -0.5 mM. This is shown in Table2.
  • NA011 Guanosine-5'- ⁇ , ⁇ - methylene triphosphate
  • NA014 is a dGTP analog with a modification on the phosphate moiety in which the oxygen atom linking the ⁇ and ⁇ phosphate groups is replaced with a methylene group.
  • NA014 demonstrated a concentration-dependent inhibition of human 293 cells telomerase activity.
  • NA013 demonstrated a concentration-dependent inhibition of human 293 cell telomerase activity.
  • NA020 is a dGTP analog in which the 2'-deoxyribose moiety is replaced with a 2',3'-dideoxyribose moiety.
  • NA020 completely at concentrations as low as 12.5 ⁇ M (0.0125mM) inhibits human 293 cell telomerase activity.
  • NA022 is an example of a dGTP analog in which the heterocyclic base, guanine, is replaced with a 7-deazaguanine.
  • NA022 demonstrated concentration-dependent inhibition of human 293 cell telomerase activity.
  • NA004 2'- Deoxyadenosine-5'- ⁇ , ⁇ -methylene triphosphate (NA004) Using the Conventional Telomerase Assay.
  • NA004 is a dATP analog with a modification on the phosphate moiety in which the oxygen atom linking the ⁇ and ⁇ phosphate groups is replaced with a methylene group.
  • NA004 demonstrated a concentration-dependent inhibition of human 293 cell telomerase activity using the modified telomerase assay in which the competing radioactive nucleotides is 3.12 ⁇ M [ ⁇ 32 P]dATP.
  • NA006 demonstrates a concentration-dependent inhibition of human 293 cell telomerase activity using the modified telomerase assay in which the competing radioactive nucleotides is 3.12 ⁇ M [ ⁇ 32 P]dATP.
  • NA007 is an example of a dATP analog in which the 2'-deoxyribose moiety is replaced with a 2',3'-dideoxyribose moiety.
  • NA007 demonstrated no inhibition of human 293 cells telomerase activity up to 2 mM.
  • NA006 demonstrated a concentration-dependent inhibition of human 293 cell telomerase activity using the modified telomerase assay in which the competing radioactive nucleotides is 3.12 ⁇ M [ ⁇ 32 P]dATP.
  • NA023 is an example of a dATP analog in which the heterocyclic base, adenine, was replaced with a 7-deazaadenine.
  • NA023 demonstrated concentration-dependent inhibition of human 293 cell telomerase activity.
  • telomerase catalyzes the polymerization of a (TTAGGG)3 primer using dTTP, dATP, dGTP.
  • TTAGGG dTTP, dATP, dGTP.
  • dTTP, dATP, and dGTP native nucleotides
  • incorporation of these nucleotide analogs leads to a prematurely shorten telomere.
  • NA004 a dATP analog with a 5'- ⁇ , ⁇ -methylene group, is able to replace dATP and become incorporated into telomeric ladders.
  • NA004 was shown to be incorporated at all the concentrations tested (0.125 - 2 mM), see FIG. 10.
  • NA006 2'-Deoxyadenosine-5'-O-(1-Thio-triphosphate) (NA006) by Human Cancer Telomerase.
  • NA006 was shown to be incorporated at the concentrations tested (0.125 - 2 mM), see FIG. 11.
  • telomere assay NA007 Using the conventional telomerase assay NA007, a dATP analog lacking the 3'-OH group, showed no apparent NA007 incorporation was detected at the concentrations tested (0.125 - 2 mM), see FIG. 12.
  • NA013 was shown to be incorporated at concentrations of 0.5 - 2 mM, see FIG. 13.
  • NA014 a dGTP analog with a 5'- ⁇ , ⁇ -methylene group, replaced dGTP and became incorporated into telomeric ladders.
  • NA014 incorporated into telomere could be detected at concentrations of 0.5-2mM, see FIG. 14.
  • NA020 a dGTP analog lacking the 3'-OH group, once incorporated into telomere was shown to chain-terminate the reaction.
  • modified telomerase assay no apparent NA020 incorporation was detected at all concentrations tested (0.125 - 2 mM), see FIG. 15.
  • telomere assay 7-deaza-dATP was replaced by 7-deaza-dATP (NA023) with 1 mM TTP and 1.56 ⁇ M [ ⁇ - 32 P]dGTP in the conventional telomerase assay).
  • the products were sensitive to RNase A and processivity decreased with increasing 7-deaza-dATP concentrations.
  • No telomerase activity was detected at > 1.5 mM 7-deaza-dATP.
  • the level of telomerase activity remained unchanged with 0.25-2 mM dATP as a substrate.
  • the presence of 7-deaza-dATP did not result in a change in pause sites compared to the reactions in the presence of dATP.
  • Examples 1-4 demonstrated various dTTP, dATP, and dGTP analogs useful for modulating human tumor telomerase activity either by inhibiting the enzyme activity or by incorporating into telomere and as the result of incorporation affecting subsequent telomere elongation.
  • the following examples demonstrate that these compounds have growth inhibitory activity.
  • phosphates mono-, di-, or triphosphates
  • the phosphates are highly negatively charged, and therefore do not effectively transport into cells. This is due to the extracellular dephosphorylation of the mono-, di-, or triphosphates by alkaline phosphorylase or 5'-nucleotidase back to the nucleoside.
  • the following table shows the growth inhibition of the indicated 7-deaza-2'- deoxynucleoside compounds against transformed human embryonic kidney cells.
  • the following table shows growth inhibition of the indicated 7-deaza-2'- deoxynucleoside compounds against human colon carcinoma cells.
  • telomeres were measured by digesting the chromosomal ends with MSEI. The telomeric fragments were separated by electrophoresis, probed for telomeric sequence and the average size of the signals produced was determined by autoradiography and densitometry. Without telomerase activity, the HeLa cells can no longer maintain their telomeres and therefore the telomeres shorten with each generation. The decrease in telomer length with cell divisions is shown below.
  • AZT AZT induced a AZT concentration dependent and cell doubling dependent destabilization of the chromosomes by inhibition of telomerase as illustrated in the graph below.
  • oligodeoxyribonucleotide of the sequence d(GGTTAGG*GTTAG) where G* represents the 7-deaza-2'-deoxyguanosine residue results in the inability of this sequence to form G-quartet structure in Kcl-containing buffers at temperaqtures above about 20°C, as evidenced by the lack of imino proton resonances in the region of 10-12 ppm in the NMR spectrum of this sample (FIG. 9).
  • the corresponding natural sequence d(GGTTAGGGTTAG) forms G-quartet structures at temperatures as high as 70°C, as evidence by the imino proton resonances at 10-12 ppm observed in the NMR spectrum of this DNA (FIG. 8).
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefened embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Balzarini et ⁇ l. "Differential patterns of intracellular metabolism of 2',3'-didehydro-2',3'- dideoxythymidine and 3'-azido-2',3'-dideoxythymidine, two potent anti-human immvmodeficiency virus compounds," J. Biol. Chem., 264:6127-6133, 1989.
  • Balzarini et ⁇ l. "The in vitro and in vivo anti-retrovirus activity, and intracellular metabolism of 3'-azido-2'3'-dideoxythymidine and 2',3'-dideoxycytidine are highly dependent on the cell species," Biochem. Pharmacol, 37:897-903, 1988.
  • telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity
  • Cell, 51:887-898, 1987 The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity
  • Harley et ⁇ l. "Telomeres shorten during aging of human fibroblasts," Nature, 345:458- 60, 1990. Harley, C. B., J. NIH Research, 7:64-68, 1995.
  • Heath et ⁇ l. "Liposome-mediate delivery of pteridine antifolates to cells in vitro: potency of methotrexate, and its a and y substituents," Biochem. Biophys. Acta,
  • McClintock "The stability of broken ends of chromosome in Zea mays, Genetics, 26:234-282, 1941. McClintock, "The fusion of broken ends of chromosomes following nuclear fusion,"
  • telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats
  • Muller et ⁇ l. "Laboratory methods: Efficient transfection and expression of heterologous genes in PC 12 cells," DNA Cell Biol, 9(3) :221-229, 1990.

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Abstract

On a découvert que des cellules souches humaines normales produisent une activité régulée de télomérase non processive alors que des cellules cancéreuses produisent une activité de télomérase processive. On a découvert que des analogues de nucléotides, tels que 7-déaza-2'-déoxyquanosine-5'-triphosphate (7-déaza-dGTP), constituent des substrats de télomerase processive et on les a incorporés dans une séquence télomère. L'incorporation de ce nucléotide a ensuite affecté la processivité de la télomérase en faisant devenir non processive une télomérase processive. On a aussi constaté que cette incorporation inhibe la formation de quadruplets G par cette séquence télomère. D'autres procédés de conversion de télomérases processives propres au cancer en des télomérases non processives plus bénignes consistent à couper partiellement l'ARN de télomérase. On a constaté que ces analogues de nucléosides pouvaient présenter différentes activités y compris médier l'inhibition, semblable à celle d'un allostère, de la télomérase, l'achèvement et le raccourcissement prématurés de l'ADN du télomère, la déstabilisation de la structure et des fonctions du télomère et finalement la mort cellulaire. Comprendre les mécanismes de modulation de la télomérase par les 7-déaza-nucléotides a permis de concevoir de nouveaux inhibiteurs ou modulateurs de la télomérase et des agents affectant la structure et les fonctions du télomère, ces découvertes étant applicables au traitement du cancer.
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US6004939A (en) * 1995-07-06 1999-12-21 Ctrc Research Foundation Board Of Regents Methods for modulation and inhibition of telomerase
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US7390891B1 (en) 1996-11-15 2008-06-24 Amgen Inc. Polynucleotides encoding a telomerase component TP2
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